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Abstract

The present study aimed to evaluate the effects of aquaporin‑1 (AQP1) level and intratumoral microvessel density (IMD) on the clinicopathological features of patients with hepatocellular carcinoma (HCC). The AQP1 expression levels, IMD and AQP1/IMD ratios in patients with HCC were measured using a semi‑quantitative immunohistochemical technique. The association between these features and clinicopathological variables were evaluated. The prognostic impact of AQP1 and IMD on overall survival (OS), and 5‑year disease‑free survival (DFS) of HCC patients was investigated retrospectively. P<0.05 was considered to indicate a statistically significant difference. A total of 90 cases of HCC were included in the present study. AQP1 was markedly expressed in the membranes of microvessels and small vessels, but seldom in hepatocellular carcinoma cells. Blood vessels in the tumors were markedly stained by anti‑cluster of differentiation 34 antibody. AQP1 expression and IMD was significantly correlated with tumor size, histologic grade, Child‑Pugh classification, microvascular invasion and tumor‑node‑metastasis (TNM) stage (P<0.05). Concurrently, for the 5‑year DFS and OS, a larger tumor size, poorly differentiated histological grade, B and C Child‑Pugh classification, presence of microvascular invasion, high TNM stage, a high AQP1 expression and a high IMD were significant risk factors for mortality. Multivariate analysis revealed that TNM stage and IMD were independent unfavorable prognostic markers for 5‑year DFS (P=0.049 and P=0.025, respectively) and OS (P=0.043 and P=0.042, respectively). These data suggest that high AQP1 expression and IMD are associated with tumor progression and prognosis in HCC. The IMD level may serve as an independent indicator for the 5‑year DFS and OS.

Introduction

It is well-known that hepatocellular carcinoma (HCC)
is one of the most common types of malignancy in South-East Asia
(1). For localized tumors, effective
treatments include surgical resection, local ablation therapy,
trans-catheter arterial chemoembolization and liver transplantation
(1–3).
However, HCC is typically diagnosed at the advanced stages in
numerous patients. Although several molecular targeting drugs have
previously been used in a clinical setting, their effects are
limited (4). Therefore, novel
molecular targets are required to manage HCC progression.

HCC is a typical hypervascular tumor, as
demonstrated by dynamic computerized tomography or angiography
(5,6),
and its progression is markedly associated with active
neovascularization (7–9). Angiogenesis is important to tumor
metastasis and growth, as it provides the oxygen, and nutrients for
tumor cells (10,11). Intratumoral microvessel density (IMD),
the most common indicator of tumor angiogenesis, is assessed using
cluster of differentiation (CD)31, CD34 or von Willebrand factor
(vWF) staining (12). It has been
suggested that an increased IMD is a predictor for decreased
disease-free survival (DFS) and overall survival (OS) rates, and
several antiangiogenic agents have begun to be used in the
treatment of HCC (9,13). However, conflicting results have also
identified that a low IMD is a significant unfavorable prognostic
factor of 2-year DFS as well as OS rate (14).

Aquaporins (AQPs) are a family of transmembrane
water channel proteins, which are expressed in numerous types of
fluid-transporting tissue, including glandular epithelia and kidney
tubules, and in non-fluid-transporting tissue, including the
epidermis. There are more than 10 AQPs that have been identified in
mammals (15). Their localization in
the plasma membrane is essential in the regulation of water
transfer (16). The first member to
be identified, AQP1, is a membrane protein that regulates the
permeability of endothelial and epithelial barriers by facilitating
water movement across cell membranes (17). In addition to its basic function,
human AQP1 expression has been revealed to be heterogeneously
expressed in different human tumors (18–23).
Several studies have identified that the upregulation of AQP1
occurs in various malignancies, including in glial tumors (18), breast cancer (19) and colorectal cancer (20). Furthermore, previous studies have
investigated AQP1 expression in the microvessels of multiple
tumors, indicating the potential involvement of AQP1 in tumor
angiogenesis (17,21). Impaired tumor angiogenesis and tumor
migration were identified in AQP1 knockout mice (22). Conversely, AQP1 overexpression is
consistent with bone marrow angiogenesis in patients with active
multiple myeloma, suggesting AQP1 is an indicator of angiogenesis
(23).

However, the role of AQPs in HCC is poorly
characterized. In the present study, the protein expression of AQP1
in HCC tissue samples was investigated, and the clinicopathological
and prognostic value of AQP1 in HCC was analyzed.

Materials and methods

Tissue specimens and clinical
data

Tumor samples and adjacent liver tissues were
collected from 90 patients with HCC who underwent curative surgical
resection without any prior anticancer therapy between May 2007 and
May 2012 at the Centre for Liver Disease in the 458th Hospital of
People's Liberation Army (Guangzhou, China). Patients with
concurrent second primary cancer were excluded. The present study
was approved by the Human Research Ethics Committee of the 458th
Hospital of People's Liberation Army, and written informed consent
was obtained from each patient. OS and DFS were defined as the
interval between dates of surgery and mortality, and between dates
of surgery and recurrence, respectively. Those patients who
developed recurrence were treated with repeated hepatic resection,
trans-catheter arterial embolization or radiofrequency ablation.
Demographical and clinicopathological data consisted of age, sex,
presence of cirrhosis, status of hepatitis B surface antigen
(HBsAg), levels of preoperative α-fetoprotein (AFP), tumor size,
histological grade, Child-Pugh classification, microvascular
invasion and tumor-node-metastasis (TNM) stage (14,24–26).
Serial sections (5 µm thick) were obtained from each tissue block,
and stained with hematoxylin and eosin (H&E; 0.2% hematoxylin
and 1% eosin) using standard pathologic procedures for 1 h.
Briefly, sections were deparaffinized in xylene (2×5 min) and
rehydrated with successive 1-min washes in 100, 96, 80 and 70%
ethanol. Sections were then stained with hematoxylin for 2 min at
room temperature, rinsed with distilled water, rinsed with 0.1%
hydrochloric acid in 50% ethanol, rinsed with tap water for 15 min,
stained with eosin for 1 min at room temperature and rinsed again
with distilled water. The slides were then dehydrated with 95 and
100% ethanol successively followed by xylene (2×5 min), and then
mounted with coverslips. H&E-stained sections were analyzed by
light microscopy (magnification, ×20) using a Leica DM LB2
epifluorescence microscope (Leica Microsystems GmbH, Wetzlar,
Germany). Images that were at the original magnification of ×20 of
H&E staining were acquired with a CCD digital camera (model
7.2; Diagnostic Instruments, Inc., Sterling Heights, MI, USA). The
mean age of the patients was 54.0±10.0 years (standard deviation;
range, 25–73). There were 73 males and 17 females. The average
tumor size was 4.4±1.8 cm (range, 1.3–7.7), with 47 tumors ≤5 cm
and 43 tumors >5 cm. Among the 90 HCC examined in the present
study, 53 exhibited hepatitis B infection. The median follow-up
time was 35.0 months.

Tissue microarray (TMA) construction
and immunohistochemistry (IHC)

Immunohistochemistry images were captured and
analyzed using Image Pro-Plus 4.5 software (Media Cybernetics,
Silver Spring, MA, USA) for integrated optical density
semi-quantitation. Leica DM LB2 epifluorescence microscope (Leica
Microsystems GmbH, Wetzlar, Germany) was used to analyze the images
of IHC at a low magnification (×100) and a high magnification
(×400). Representative sections of HCC or normal liver tissues in
the pre-existing paraffin-embedded tissue blocks were determined
according to the aforementioned H&E staining slides. The TMA
was prepared using a needle to punch a 1.5 mm diameter cylinder in
the representative section of each tissue, and by placing the
cylinders into an array on a recipient paraffin block. Sections
were cut 2-µm thick from the TMA block and mounted on microscope
slides. The TMA consisted of a total of 90 patients with HCC and 90
cases of paraffin-embedded adjacent normal tissue. The
clinicopathological characteristics of patients are summarized in
Table I. The TMA slides were dried
overnight at 37°C, dewaxed in xylene, rehydrated using an alcohol
gradient, and the endogenous peroxidase activity was blocked by
immersing the slides in 0.3% hydrogen peroxide
(H2O2) for 10 min at room temperature.
Antigen retrieval was performed through microwave heating with
sodium citrate buffer (pH 6.0) at 100°C for 30 min. Then,
non-specific binding sites were blocked at room temperature using
the blocking buffer from the Vectastain® Elite ABC kit
(Vector Laboratories, Peterborough, UK) for 45 min. Samples were
then incubated with mouse monoclonal anti-human antibody against
AQP1 (1:500 dilution; cat. no. ab9566; Abcam, Cambridge, MA, USA)
and mouse monoclonal CD34 (1:50 dilution; cat. no. MA1-10202; clone
QB End10; Neomarkers, Inc., Fremont, CA, USA) primary antibodies at
room temperature for 60 min. Following three washes with PBS, the
slides were sequentially incubated with a polymer
peroxidase-labeled rabbit anti-mouse secondary antibody (100
dilution; cat. no. ZDR-5109; ZSGB-BIO, Beijing, China) for 30 min
at room temperature. Then, the slides were stained at 37°C for 1 h
using the 3,3′-diaminobenzidine horseradish peroxidase Color
Development kit (Beyotime Institute of Biotechnology, Haimen,
China). Finally, the sections were counterstained with hematoxylin
for 5 min at room temperature. Known IHC positive slides were used
as a positive control, and anti-AQP1 primary antibody was replaced
with PBS as a negative control.

Evaluation of IMD and AQP1
expression

IMD scores were assessed by immunostaining for CD34
according to Weidner (24).
Subsequent to scanning the immunostained section at a low
magnification (×100), the area within the tumor or adjacent tissues
with the highest number of distinctly highlighted microvessels was
selected as the ‘hot spot’. IMD was defined by the mean value of
vessel number visualized at high magnification (×400) in five
fields within the hot spot. Evaluation of the staining reactions
was strictly confined to the area of highest IMD. For the
sinusoid-like microvessels, which were primarily observed in the
areas with a large trabecular structure and assessed using a
modified method introduced by Tanigawa et al (27), every 40-µm length of lumen was counted
as 1 point. Each stained lumen was regarded as a single countable
microvessel. If there was no lumen, but only a single positive cell
was visible, this cell was also interpreted as representing a
microvessel. Any positive staining of endothelium or mass of
endothelium clearly separated from the surrounding tumor cells and
connective tissue was counted as a microvessel. Immunohistochemical
analysis was performed independently by two investigators (Dr
Li-Min Luo, 458th Hospital of People's Liberation Army and Dr Min
Wei, Southern Medical University). The mean values were accepted if
the two investigators agreed with the values. If the differences
between the observers were >30%, the values were re-estimated
until a consensus was reached. The expression of AQP1 was detected
and assessed using the same method. As the number of microvessels
observed may vary by patient and vascular spots, resulting in an
error in any measurement of AQP1 expression, the AQP1/IMD ratio was
also assessed to avoid this error.

Statistical analysis

Statistical analysis was performed using SPSS
software (version 18.0; SPSS, Inc., Chicago, USA). The associations
between clinical and prognostic variables (patient age, sex,
cirrhosis, HBsAg, AFP, tumor size, histological grade, Child-Pugh
classification, microvascular invasion and TNM stage, and AQP1
expression, IMD and the AQP1/IMD ratio) were determined. Un-paired
Student's t-tests were used to compare values between two groups,
and one-way analysis of variance was performed when ≥3 groups were
present. Correlations were determined by Spearman rank correlation
test. A two-tailed P<0.05 was considered to indicate a
statistically significant difference.

Results

Association between the AQP1
expression/IMD and the clinicopathological factors of the
patients

There were two types of microvessels identified:
Capillary-like microvessels with small, scattered capillaries with
no or a narrow lumen, and sinusoid-like microvessels with
continuous branching and a distinct lumen structure.
Immunohistochemical analysis demonstrated that the AQP1 protein was
markedly expressed in the membrane of microvessels and small
vessels in the majority of HCC samples (Fig. 1A and B), but seldom in the cytoplasm
of tumor cells. The AQP1 expression in microvessels of HCC
presented a significant association with cirrhosis, tumor size,
histological grade, Child-Pugh classification, microvascular
invasion and TNM stage. The expression of AQP1 was significantly
higher in the presence of cirrhosis compared with in absence of
cirrhosis (P=0.048), in tumor sizes >5 cm compared with in tumor
sizes ≤5 cm (P<0.001), in poorly differentiated histological
grades compared with in well or moderately differentiated
histological grade (P=0.001), in Child-Pugh classification B + C
compared with in Child-Pugh classification A (P=0.007), in the
presence of microvascular invasion compared with in absence of
microvascular invasion (P<0.001) and in TNM stage III–IV
compared with in TNM stage I–II (P<0.001) (Table II). However, no significant
variations according to HBsAg and AFP levels were observed
(P>0.05; Table II). CD34 was also
highly expressed in the membrane of microvessels and small vessels
in the majority of HCC samples (Fig. 1C
and D). The IMD score, assessed by CD34 immunostaining, was
significantly associated with tumor size, histological grade,
Child-Pugh classification, microvascular invasion and TNM stage.
IMD scores were higher in tumor sizes >5 cm compared with in
tumor sizes ≤5 cm (P<0.001), in poorly differentiated
histological grade compared with in well and moderately
differentiated histological grades (P=0.002), in Child-Pugh
classification B + C compared with in Child-Pugh classification A
(P=0.019), in the presence of microvascular invasion compared with
in absence of microvascular invasion (P<0.001) and in TNM stage
III–IV compared with in TNM stage I–II (P<0.001) (Table II). However, no significant
differences between IMD score and cirrhosis, HBsAg or AFP were
observed (P>0.05; Table II). A
statistically significant positive correlation was observed between
AQP1 expression and the IMD scores (r=0.227; P<0.001).

Discussion

The ability of tumor cells to grow and migrate
requires a sufficient blood supply. A number of malignant tumors
have been identified to induce neovascularization (28,29).
Tanigawa et al (27)
demonstrated an increased microvessel density in malignant HCC and
indicated that IMD was a prognostic factor for HCC. However, the
clinicopathological significance of angiogenesis in HCC remains to
be elucidated (13,14,30). Due
to the diversities in tissue processing and immunostaining
techniques, including the observation for selected vascular hot
spots, antibodies to identify endothelial cells, and the method of
counting the vessels, the results of angiogenesis are not able to
be corroborated easily. Anti-CD34 antibodies have been identified
to be better at identifying endothelial cells compared with
anti-CD31 and anti-vWF antibodies, and with greater sensitivity
(13,14,27). The
anti-CD34 antibody has been suggested to be the most sensitive and
specific marker among the other endothelial markers in HCC
(31). Therefore, IMD score was
assessed using the anti-CD34 antibody in the present study. The
results suggest that IMD may serve an important role in the HCC due
to its association with tumor size, histological grade, Child-Pugh
classification, microvascular invasion and TNM stage, which was in
accordance with Tanigawa et al (27), and Wang et al (32), who hypothesized that IMD is an
independent prognostic factor for HCC.

The number of microvessels in tumors varies in
different patients or hot spots, which may result in differences in
AQP1 expression measurements. For IMD, defined as tumor microvessel
counts, and AQP1 protein, which is primarily expressed in
microvessels, the AQP1/IMD ratio may determine the association
between IMD and AQP1 expression levels in the microvessels of HCC,
and correct subjective and objective errors. In the present study,
AQP1 protein was highly expressed in the membranes of microvessels
and small vessels within the majority of patients with HCC, but was
expressed seldom in the cytoplasm of the tumor cells. The
distribution of AQP1 protein indicated that AQP1 may serve an
important role in transvascular water transport in primary HCC, and
exhibits little effect on water flow in tumor cells.

The expression of AQP1 in HCC tissues was higher
compared with that of adjacent normal liver tissues. These data
indicate the potential role of AQP1 during HCC carcinogenesis. It
is possible that the induction of AQP1 is required in the
development of HCC and serves as an essential driving force for
initiating carcinogenesis. During the cell cycle, as the cell
volume needs to expand rapidly by absorbing water from the
extracellular environment with a minimal volume of energy,
upregulation of AQP1 in microvessels is potentially advantageous
for the growth or survival of tumor cells (33). Furthermore, the result suggests that
HCC, similar to other solid tumors, exhibit high vascular
permeability (34).

Previous studies have demonstrated that AQP1
expression is upregulated in astrocytomas and metastatic carcinomas
(35,36), and AQP1 expression in the microvessels
of neoplastic brain cells was proposed to increase blood-brain
barrier water permeability, resulting in brain tumor edema in
aggressive brain tumors (37).

In addition, the results of the present study
indicated that AQP1 expression in the microvessels of HCC samples
was significantly associated with tumor size, histologic grade,
Child-Pugh classification, microvascular invasion and TNM stage.
The survival analysis results suggested that the AQP1 protein may
be upregulated in the advanced stages of the disease, and may be
involved in the progression and prognosis of HCC.

In the present study, Spearman correlations
demonstrated that there was a positive correlation between IMD and
the expression of AQP1. These results suggest that AQP1 expression
in microvessel endothelial cells of HCC may be associated with
angiogenesis. Additional experiments are required to investigate
whether AQP1 overexpression or knockout in tumor microvessels
affect angiogenesis directly.

Papadopoulos and Verkman (38) demonstrated that the pharmacological
modulation of AQP1 function may provide novel therapeutic
approaches in human disease, including diuretics, and regulators of
intraocular pressure and swelling in the brain, and cornea. In
addition, Ma et al (39)
suggested that topiramate decreases AQP1 protein immunostaining in
lung carcinoma microvessel endothelial cells of mice, and
hypothesized that the suppression of AQP1 expression may be an
important factor for the inhibitory action of topiramate on tumor
metastasis. In conclusion, the results of the present study
indicate that high AQP1 expression may serve an essential role in
HCC carcinogenesis and progression. Additional studies
investigating the molecular mechanisms of AQP1 regulation, and the
association between AQP1 expression and tumor angiogenesis, are
required to verify this novel therapy for HCC.

Acknowledgements

The present study was supported by the National
Natural Science Foundation of China (grant no. 81672754), the
Natural Science Foundation of Guangdong province (grant no.
2015A030313249) and the ‘Twelfth Five Year Plan’ research project,
Medical Department of General Logistics Department of China (grant
no. CWS11J021).